Deletion of VPS50 protein in mouse brain impairs synaptic function and behavior.

BACKGROUND: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. RESULTS: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. CONCLUSIONS: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.

Skeletal myotubes expressing ALS mutant SOD1 induce pathogenic changes, impair mitochondrial axonal transport, and trigger motoneuron death.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons (MNs), and despite progress, there is no effective treatment. A large body of evidence shows that astrocytes expressing ALS-linked mutant proteins cause non-cell autonomous toxicity of MNs. Although MNs innervate muscle fibers and ALS is characterized by the early disruption of the neuromuscular junction (NMJ) and axon degeneration, there are controversies about whether muscle contributes to non-cell-autonomous toxicity to MNs. In this study, we generated primary skeletal myotubes from myoblasts derived from ALS mice expressing human mutant SOD1 G93A (termed hereafter mutSOD1). Characterization revealed that mutSOD1 skeletal myotubes display intrinsic phenotypic and functional differences compared to control myotubes generated from non-transgenic (NTg) littermates. Next, we analyzed whether ALS myotubes exert non-cell-autonomous toxicity to MNs. We report that conditioned media from mutSOD1 myotubes (mutSOD1-MCM), but not from control myotubes (NTg-MCM), induced robust death of primary MNs in mixed spinal cord cultures and compartmentalized microfluidic chambers. Our study further revealed that applying mutSOD1-MCM to the MN axonal side in microfluidic devices rapidly reduces mitochondrial axonal transport while increasing Ca2+ transients and reactive oxygen species (i.e., H 2 O 2 ). These results indicate that soluble factor(s) released by mutSOD1 myotubes cause MN axonopathy that leads to lethal pathogenic changes.

Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

BACKGROUND: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. RESULTS: Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700 bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300 bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD. CONCLUSIONS: Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

Deletion of VPS50 protein in mice brain impairs synaptic function and behavior.

VPS50, is an accessory protein, involved in the synaptic and dense core vesicle acidification and its alterations produce behavioral changes in C.elegans. Here, we produce the mosaic knock out (mKO) of VPS50 using CRISPR/Cas9 system in both cortical cultured neurons and whole animals to evaluate the effect of VPS50 in regulating mammalian brain function and behavior. While mKO of VPS50 does not change the number of synaptic vesicles, it produces a mislocalization of the V-ATPase pump that likely impact in vesicle acidification and vesicle content to impair synaptic and neuronal activity in cultured neurons. In mice, mKO of VPS50 in the hippocampus, alter synaptic transmission and plasticity, and generated robust cognitive impairments associate to memory formation. We propose that VPS50 is an accessory protein that aids the correct recruitment of the V-ATPase pump to synaptic vesicles, thus having a crucial role controlling synaptic vesicle acidification and hence synaptic transmission.

Removal of a genomic duplication by double-nicking CRISPR restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. Using the ASD and anxiety mouse model Flailer, that contains a duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700bp genomic duplication in vitro and in vivo . Importantly, DN-CRISPRs have not been used to remove more gene regions <100bp successfully and with high efficiency. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene edition. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescues some of the mutant behaviors, while intracerebroventricular delivery, completely recovers Flailer animal phenotype associated to anxiety and ASD. Our results demonstrate the potential of DN-CRISPR to efficiently (>60% editing in vivo) remove large genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

Myosin Va Brain-Specific Mutation Alters Mouse Behavior and Disrupts Hippocampal Synapses.

Myosin Va (MyoVa) is a plus-end filamentous-actin motor protein that is highly and broadly expressed in the vertebrate body, including in the nervous system. In excitatory neurons, MyoVa transports cargo toward the tip of the dendritic spine, where the postsynaptic density (PSD) is formed and maintained. MyoVa mutations in humans cause neurologic dysfunction, intellectual disability, hypomelanation, and death in infancy or childhood. Here, we characterize the Flailer (Flr) mutant mouse, which is homozygous for a myo5a mutation that drives high levels of mutant MyoVa (Flr protein) specifically in the CNS. Flr protein functions as a dominant-negative MyoVa, sequestering cargo and blocking its transport to the PSD. Flr mice have early seizures and mild ataxia but mature and breed normally. Flr mice display several abnormal behaviors known to be associated with brain regions that show high expression of Flr protein. Flr mice are defective in the transport of synaptic components to the PSD and in mGluR-dependent long-term depression (LTD) and have a reduced number of mature dendritic spines. The synaptic and behavioral abnormalities of Flr mice result in anxiety and memory deficits similar to that of other mouse mutants with obsessive-compulsive disorder and autism spectrum disorder (ASD). Because of the dominant-negative nature of the Flr protein, the Flr mouse offers a powerful system for the analysis of how the disruption of synaptic transport and lack of LTD can alter synaptic function, development and wiring of the brain and result in symptoms that characterize many neuropsychiatric disorders.

Synaptic Effects of Dopamine Breakdown and Their Relation to Schizophrenia-Linked Working Memory Deficits.

Working memory is the ability to hold information "online" over a time delay in order to perform a task. This kind of memory is encoded in the brain by persistent neural activity that outlasts the presentation of a stimulus. Patients with schizophrenia perform poorly in working memory tasks that require the brief memory of a target location in space. This deficit indicates that persistent neural activity related to spatial locations may be impaired in the disease. At the circuit level, many studies have shown that NMDA receptors and the dopamine system are involved in both schizophrenia pathology and working memory-related persistent activity. In this Hypothesis and Theory article, we examine the possible connection between NMDA receptors, the dopamine system, and schizophrenia-linked working memory deficits. In particular, we focus on the dopamine breakdown product homocysteine (HCY), which is consistently elevated in schizophrenia patients. Our previous studies have shown that HCY strongly reduces the desensitization of NMDA currents. Here, we show that HCY likely affects NMDA receptors in brain regions that support working memory; this is because these areas favor dopamine breakdown over transport to clear dopamine from synapses. Finally, within the context of two NMDA-based computational models of working memory, we suggest a mechanism by which HCY could give rise to the working memory deficits observed in schizophrenia patients.

The Conserved VPS-50 Protein Functions in Dense-Core Vesicle Maturation and Acidification and Controls Animal Behavior.

The modification of behavior in response to experience is crucial for animals to adapt to environmental changes. Although factors such as neuropeptides and hormones are known to function in the switch between alternative behavioral states, the mechanisms by which these factors transduce, store, retrieve, and integrate environmental signals to regulate behavior are poorly understood. The rate of locomotion of the nematode Caenorhabditis elegans depends on both current and past food availability. Specifically, C. elegans slows its locomotion when it encounters food, and animals in a food-deprived state slow even more than animals in a well-fed state. The slowing responses of well-fed and food-deprived animals in the presence of food represent distinct behavioral states, as they are controlled by different sets of genes, neurotransmitters, and neurons. Here we describe an evolutionarily conserved C. elegans protein, VPS-50, that is required for animals to assume the well-fed behavioral state. Both VPS-50 and its murine homolog mVPS50 are expressed in neurons, are associated with synaptic and dense-core vesicles, and control vesicle acidification and hence synaptic function, likely through regulation of the assembly of the V-ATPase complex. We propose that dense-core vesicle acidification controlled by the evolutionarily conserved protein VPS-50/mVPS50 affects behavioral state by modulating neuropeptide levels and presynaptic neuronal function in both C. elegans and mammals.

A Diencephalic Dopamine Source Provides Input to the Superior Colliculus, where D1 and D2 Receptors Segregate to Distinct Functional Zones.

Modulation of neural responses is frequently observed in the superior colliculus (SC), a retinorecipient midbrain structure that controls orienting and the localization of attention. Although behavioral contingencies that influence SC responses are well documented, the neural pathways and molecular mechanisms responsible for this modulation are not completely understood. Here, we illustrate a dopaminergic system that strongly impacts neural responses in the SC. After using RNA sequencing (RNA-seq) to detail the transcriptome of dopamine-related genes in the SC, we show that D1 receptors are enriched in the superficial visual SC, while D2 receptors segregate to the intermediate multimodal/motor SC. Retrograde injections into the SC consistently label A13, a small dopamine cell group located in the zona incerta. We surmise that A13 mimics dopaminergic effects that we observed in SC slices, which suggests that dopamine in the SC may reduce the tendency of an animal to orient or attend to salient stimuli.

PSD95 suppresses dendritic arbor development in mature hippocampal neurons by occluding the clustering of NR2B-NMDA receptors.

Considerable evidence indicates that the NMDA receptor (NMDAR) subunits NR2A and NR2B are critical mediators of synaptic plasticity and dendritogenesis; however, how they differentially regulate these processes is unclear. Here we investigate the roles of the NR2A and NR2B subunits, and of their scaffolding proteins PSD-95 and SAP102, in remodeling the dendritic architecture of developing hippocampal neurons (2-25 DIV). Analysis of the dendritic architecture and the temporal and spatial expression patterns of the NMDARs and anchoring proteins in immature cultures revealed a strong positive correlation between synaptic expression of the NR2B subunit and dendritogenesis. With maturation, the pruning of dendritic branches was paralleled by a strong reduction in overall and synaptic expression of NR2B, and a significant elevation in synaptic expression of NR2A and PSD95. Using constructs that alter the synaptic composition, we found that either over-expression of NR2B or knock-down of PSD95 by shRNA-PSD95 augmented dendritogenesis in immature neurons. Reactivation of dendritogenesis could also be achieved in mature cultured neurons, but required both manipulations simultaneously, and was accompanied by increased dendritic clustering of NR2B. Our results indicate that the developmental increase in synaptic expression of PSD95 obstructs the synaptic clustering of NR2B-NMDARs, and thereby restricts reactivation of dendritic branching. Experiments with shRNA-PSD95 and chimeric NR2A/NR2B constructs further revealed that C-terminus of the NR2B subunit (tail) was sufficient to induce robust dendritic branching in mature hippocampal neurons, and suggest that the NR2B tail is important in recruiting calcium-dependent signaling proteins and scaffolding proteins necessary for dendritogenesis.

Postsynaptic localization of PSD-95 is regulated by all three pathways downstream of TrkB signaling.

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB regulate synaptic plasticity. TrkB triggers three downstream signaling pathways; Phosphatidylinositol 3-kinase (PI3K), Phospholipase Cγ (PLCγ) and Mitogen activated protein kinases/Extracellular signal-regulated kinases (MAPK/ERK). We previously showed two distinct mechanisms whereby BDNF-TrkB pathway controls trafficking of PSD-95, which is the major scaffold at excitatory synapses and is critical for synapse maturation. BDNF activates the PI3K-Akt pathway and regulates synaptic delivery of PSD-95 via vesicular transport (Yoshii and Constantine-Paton, 2007). BDNF-TrkB signaling also triggers PSD-95 palmitoylation and its transport to synapses through the phosphorylation of the palmitoylation enzyme ZDHHC8 by a protein kinase C (PKC; Yoshii etal., 2011). The second study used PKC inhibitors chelerythrine as well as a synthetic zeta inhibitory peptide (ZIP) which was originally designed to block the brain-specific PKC isoform protein kinase Mϖ (PKMϖ). However, recent studies raise concerns about specificity of ZIP. Here, we assessed the contribution of TrkB and its three downstream pathways to the synaptic distribution of endogenous PSD-95 in cultured neurons using chemical and genetic interventions. We confirmed that TrkB, PLC, and PI3K were critical for the postsynaptic distribution of PSD-95. Furthermore, suppression of MAPK/ERK also disrupted PSD-95 expression. Next, we examined the contribution of PKC. While both chelerythrine and ZIP suppressed the postsynaptic localization of PSD-95, RNA interference for PKMϖ did not have a significant effect. This result suggests that the ZIP peptide, widely used as the "specific" PKMϖ antagonist by many investigators may block a PKC variant other than PKMϖ such as PKCλ/ι. Our results indicate that TrkB regulates postsynaptic localization of PSD-95 through all three downstream pathways, but also recommend further work to identify other PKC variants that regulate palmitoylation and synaptic localization of PSD-95.

Independent optical excitation of distinct neural populations.

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

Homocysteine reduces NMDAR desensitization and differentially modulates peak amplitude of NMDAR currents, depending on GluN2 subunit composition.

N-methyl-d-aspartate receptors (NMDARs) have been linked to schizophrenia because agents that bind the receptor, like ketamine and phencyclidine, are capable of inducing schizophrenia-like symptoms. Here we show that the amino acid homocysteine (HCY), which is increased in the blood of schizophrenia patients, reduces desensitization of NMDARs in cultured mouse neurons, human embryonic kidney cells transfected with GluN1 + GluN2A, GluN2B, or GluN2D subunits, and hippocampal slices. HCY also alters the peak amplitude of NMDAR currents, depending on the GluN2 subunit the receptor contains; GluN1 + GluN2A-containing NMDARs show an increase in peak amplitude when exposed to HCY, while GluN1 + GluN2B-containing NMDARs show a decrease in peak amplitude. Both peak amplitude and desensitization effects of HCY can be occluded by saturating the NMDAR with glycine. Since glycine concentrations are not saturating in the brain, HCY could play an NMDAR-modulating role in the nervous system. We also show that HCY shares characteristics with glutamate and suggest that HCY affects both the agonist and co-agonist site of the NMDAR.

A Myosin Va mutant mouse with disruptions in glutamate synaptic development and mature plasticity in visual cortex.

Myosin Va (MyoVa) mediates F-actin-based vesicular transport toward the plasma membrane and is found at neuronal postsynaptic densities (PSDs), but the role of MyoVa in synaptic development and function is largely unknown. Here, in studies using the dominant-negative MyoVa neurological mutant mouse Flailer, we find that MyoVa plays an essential role in activity-dependent delivery of PSD-95 and other critical PSD molecules to synapses and in endocytosis of AMPA-type glutamate receptors (AMPAR) in the dendrites of CNS neurons. MyoVa is known to carry a complex containing the major scaffolding proteins of the mature PSD, PSD-95, SAPAP1/GKAP, Shank, and Homer to dendritic spine synapses. In Flailer, neurons show abnormal dendritic shaft localization of PSD-95, stargazin, dynamin3, AMPARs and abnormal spine morphology. Flailer neurons also have abnormally high AMPAR miniature current frequencies and spontaneous AMPAR currents that are more frequent and larger than in wild-type while numbers of NMDAR containing synapses remain normal. The AMPAR abnormalities are consistent with a severely disrupted developmental regulation of long-term depression that we find in cortical Flailer neurons. Thus MyoVa plays a fundamentally important role both in localizing mature glutamate synapses to spines and in organizing the synapse for normal function. For this reason Flailer mice will be valuable in further dissecting the role of MyoVa in normal synaptic and circuit refinement and also in studies of neurological and neuropsychiatric diseases where disruptions of normal glutamate synapses are frequently observed.

Postsynaptic density scaffold SAP102 regulates cortical synapse development through EphB and PAK signaling pathway.

Membrane-associated guanylate kinases (MAGUKs), including SAP102, PSD-95, PSD-93, and SAP97, are scaffolding proteins for ionotropic glutamate receptors at excitatory synapses. MAGUKs play critical roles in synaptic plasticity; however, details of signaling roles for each MAGUK remain largely unknown. Here we report that SAP102 regulates cortical synapse development through the EphB and PAK signaling pathways. Using lentivirus-delivered shRNAs, we found that SAP102 and PSD-95, but not PSD-93, are necessary for excitatory synapse formation and synaptic AMPA receptor (AMPAR) localization in developing mouse cortical neurons. SAP102 knockdown (KD) increased numbers of elongated dendritic filopodia, which is often observed in mouse models and human patients with mental retardation. Further analysis revealed that SAP102 coimmunoprecipitated the receptor tyrosine kinase EphB2 and RacGEF Kalirin-7 in neonatal cortex, and SAP102 KD reduced surface expression and dendritic localization of EphB. Moreover, SAP102 KD prevented reorganization of actin filaments, synapse formation, and synaptic AMPAR trafficking in response to EphB activation triggered by its ligand ephrinB. Last, p21-activated kinases (PAKs) were downregulated in SAP102 KD neurons. These results demonstrate that SAP102 has unique roles in cortical synapse development by mediating EphB and its downstream PAK signaling pathway. Both SAP102 and PAKs are associated with X-linked mental retardation in humans; thus, synapse formation mediated by EphB/SAP102/PAK signaling in the early postnatal brain may be crucial for cognitive development.

Eye opening and PSD95 are required for long-term potentiation in developing superior colliculus.

The only major glutamate receptor membrane-associated guanylate kinase scaffolds expressed in the young superficial superior colliculus (SC) are synapse-associated protein 102 (SAP102) and postsynaptic density protein 95 (PSD95). In this, as in all visual brain regions examined, synaptic PSD95 increases rapidly following simultaneous eyelid opening (EO). We show that EO and PSD95 are necessary for SC NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) and this LTP is eliminated or reinstated by manipulating EO. PSD95 knockdown (KD) in vivo blocks this LTP, but not long-term depression, and reduces frequencies of miniature AMPA receptor and NMDAR currents with no change in presynaptic release. Furthermore, miniature NMDAR currents after PSD95 KD show an activity-triggered calcineurin sensitivity that is normally only found in the pre-EO period when SAP102 binds mixed GluN2A/GluN2B NMDARs. These data indicate that young SC LTP arises from PSD95 unsilencing of silent synapses, that unsilencing is labile in young brain, and that even though SAP102 and PSD95 can bind the same NMDARs, only PSD95 enables SC synaptic maturation.

A synaptic strategy for consolidation of convergent visuotopic maps.

The mechanisms by which experience guides refinement of converging afferent pathways are poorly understood. We describe a vision-driven refinement of corticocollicular inputs that determines the consolidation of retinal and visual cortical (VC) synapses on individual neurons in the superficial superior colliculus (sSC). Highly refined corticocollicular terminals form 1-2 days after eye-opening (EO), accompanied by VC-dependent filopodia sprouting on proximal dendrites, and PSD-95 and VC-dependent quadrupling of functional synapses. Delayed EO eliminates synapses, corticocollicular terminals, and spines on VC-recipient dendrites. Awake recordings after EO show that VC and retina cooperate to activate sSC neurons, and VC light responses precede sSC responses within intervals promoting potentiation. Eyelid closure is associated with more protracted cortical visual responses, causing the majority of VC spikes to follow those of the colliculus. These data implicate spike-timing plasticity as a mechanism for cortical input survival, and support a cooperative strategy for retinal and cortical coinnervation of the sSC.

TrkB and protein kinase Mζ regulate synaptic localization of PSD-95 in developing cortex.

Postsynaptic density 95 (PSD-95), the major scaffold at excitatory synapses, is critical for synapse maturation and learning. In rodents, eye opening, the onset of pattern vision, triggers a rapid movement of PSD-95 from visual neuron somata to synapses. We showed previously that the PI3 kinase-Akt pathway downstream of BDNF/TrkB signaling stimulates synaptic delivery of PSD-95 via vesicular transport. However, vesicular transport requires PSD-95 palmitoylation to attach it to a lipid membrane. Also, PSD-95 insertion at synapses is known to require this lipid modification. Here, we show that BDNF/TrkB signaling is also necessary for PSD-95 palmitoylation and its transport to synapses in mouse visual cortical layer 2/3 neurons. However, palmitoylation of PSD-95 requires the activation of another pathway downstream of BDNF/TrkB, namely, signaling through phospholipase Cγ and the brain-specific PKC variant protein kinase M ζ (PKMζ). We find that PKMζ selectively regulates phosphorylation of the palmitoylation enzyme ZDHHC8. Inhibition of PKMζ results in a reduction of synaptic PSD-95 accumulation in vivo, which can be rescued by overexpressing ZDHHC8. Therefore, TrkB and PKMζ, two critical regulators of synaptic plasticity, facilitate PSD-95 targeting to synapses. These results also indicate that palmitoylation can be regulated by a trophic factor. Our findings have implications for neurodevelopmental disorders as well as aging brains.